α-ketoamide inhibitors of 20S proteasome

ABSTRACT

α-ketoamide compounds useful for treating disorders mediated by 20S proteasome in mammals having the following formula: wherein X 2  is Ar or Ar--X 3  wherein X 3  is --C═O, or --CH 2  CO--, and wherein Ar is phenyl, substituted phenyl, indole, substituted indoles, and any other heteroaryls; R 1 , and R 2  are each individually selected from the side chains of the known natural α-amino acids and unnatural amino acids, hydrogen, 1-10 carbon linear and branched alkyl, 1-10 carbon linear and branched substituted alkyl, aryl, substituted aryl, 1-10 carbon linear, branched substituted aryl, alkoxyaryl, 3-8 carbon cycloalkyl, heterocycle substituted heterocycle, heteroaryl and substituted heteroaryl; X 1  is selected from hydroxide, monoalkylamino, dialkylamino, alkoxide, arylkoxide and ##STR1## wherein X 4  is hydroxide, arylamino, monoalkylamino, dialkylamino, alkoxide, or arylalkoxide; and R 3  is selected from the known natural α-amino acids, unnatural amino acids, hydrogen, 1-10 carbon linear and branched alkyl, 1-10 carbon linear and branched substituted alkyl, aryl, substituted aryl, 1-10 carbon linear and branched substituted aryl, alkoxyaryl, 3-8 carbon cycloalkyl, heterocycle, substituted heterocycle, heteroaryl and substituted heteroaryl.

BACKGROUND OF THE INVENTION

The multicatalytic proteinase or the proteasome is a highly conservedcellular structure that is responsible for the ATP-dependent proteolysisof most cellular proteins (Coux, O., Tanaka, K. and Goldberg, A. 1996Ann. Rev. Biochem. 65, 801-847). The 20S proteasome contains thecatalytic core of the complex and has been crystallized from thearchaebacteria Thermoplasma acidophilum (Lowe, J., Stock, D., Jap, B.,Zwicki, P., Bauminster, W. and Huber, R. 1995 Science 268, 533-539) andfrom the yeast Saccharomyces cerevisiae (Groll, M., Ditzel, L., Lowe,J., Stock, D., Bochtler, M., Bartunik, HD and Huber, R. 1997 Nature 386,463-471). Unlike the archaebacterial proteasome that primarily exhibitschymotrypsin-like proteolytic activity (Dahlmann, B., Kopp, F., Kuehn,L., Niedel, B., Pfeifer, G. 1989 FEBSLett. 251, 125-131; Seemuller, E.,Lupas, A., Zuw, F., Zwickl, P and Baumeister, W. FEBS Lett. 359, 173,(1995) the eukaryotic proteasome contains at least five identifiableproteolytic activities. Three of these activities are similar inspecificity to chymotrypsin, trypsin and peptidylglutamyl peptidase. Thetwo other activities described exhibit a preference for cleavage ofpeptide bonds on the carboxyl side of branched chain amino acids (BrAAP)and toward peptide bonds between short chain neutral amino acids (SnAAP)(Orlowski, M. 1990 Biochemistry 29, 10289-10297).

Although the 20S proteasome contains the proteolytic core, it cannotdegrade proteins in vivo unless it is complexed with a 19S cap, ateither end of its structure, which itself contains multiple ATPaseactivities. This larger structure is known as the 26S proteasome andwill rapidly degrade proteins that have been targeted for degradation bythe addition of multiple molecules of the 8.5-kDa polypeptide, ubiquitin(reviewed in Coux, O., Tanaka, K. and Goldberg, A. 1996 Ann.Rev.Biochem. 65, 801-847).

A large number of substrate-derived functionalities have been used aspotential serine- and thiol protease inhibitors. Several of these motifshave been described as inhibitors to the proteasome. These include thepeptide aldehydes (Vinitsky, A., Michaud, C,, Powers, J. and Orlowski,M. 1992 Biochemistry 31, 9421-9428; Tsubuki, S., Hiroshi, K., Saito, Y.,Miyashita, N., Inomata, M., and Kawashima, S. 1993Biochem.Biophys.Res.Commun. 196,1195-1201; Rock, K, I., Gramm, C.,Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D. and Goldberg,A. L. (1994) Cell 78, 761-771)N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (ALLN) andN-acetyl-L-leucinyl-1-leucinyl-methional (LLM) with the most potentinhibitor of this type beingN-carbobenzoxyl-1-L-leucinyl-L-leucinyl-L-norvalinal (MG 115). Otherreports have described a series of dipeptide inhibitors that have IC₅₀values in the 10 to 100 nM range (Iqbal, M., Chatterjee S., Kauer, J.C., Das, M., Messina, P., Freed, B., Biazzo, W and Siman, R. 1995I-Med.Chem. 38, 2276-2277). A series of α-ketocarbonyl and boronic esterderived dipeptides (Iqbai, M., Chatterjee, S., Kauer, J. C., Mallamo, J.P., Messina, P. A., Reiboldt, A. and Siman, R. 1996 Bioorg. Med-Chem.Lett 6, 287-290) and epoxyketones (Spattenstein, A., Leban, JJ., Huang,J. J., Reinhardt, K. R., Viveros, O. H., Sigafoos, J. and Crouch, R.1996 Tet. Lett. 37, 1434-1346) have also been described that are potentinhibitors of the proteasome.

A different compound that exhibits specificity in inhibiting proteasomeactivity is Lactacystin (Fenteany, G., Standaert, R. F., Lane, W. S.,Choi, S., Corey, E. J. and Schreiber, S. L. 1995 Science 268, 726-731)which is a Streptomyces metabolite. This molecule was originallydiscovered for its ability to induce neurite outgrowth in aneuroblastoma cell line (Omura, S., Matsuzaki, K., Fujimoto, T., Kosuge,K., Furuya, T., Fujita, S. and Nakagawa, A. 1991 J.Antibiot. 44,117-118) and later was shown to inhibit the proliferation of severalcell types (Fenteany, G., Standaert, R. F., Reichard, G. A., Corey, E.J. and Schreiber, S. L. 1994 Proc.Nat'l. Acad.Sci. USA 91, 3358-3362).

It is now well established that the proteasome is a major extralysosomalproteolytic system involved in the proteolytic pathways essential fordiverse cellular functions such as cell division, antigen processing andthe degradation of short-lived regulatory proteins such as oncogeneproducts, cyclins and transcription factors (Ciechanover, A. (1994) Cell79, 13-21; Palombell, V. J., Rando, O. J., Goldberg, A. L. and Maniatis,T. 1994 Cell 78, 773-785). For example, the active form of NF-κB is aheterodimer consisting of a p65 and a p50 subunit. The latter is presentin the cytosol as an inactive precursor (p105). The proteolyticprocessing of p105 to generate p50 occurs via the ubiquitin-proteasomepathway. Additionally, processed p50 and p65 are maintained in thecytosol as an inactive complex bound to the inhibitory protein IκB.Inflammatory stimuli such as LPS activate NF-κB by initiating thesignalling pathway which leads to the degradation of IκB. These signalsalso stimulate the processing of p105 into p50. Thus two proteolyticevents, both governed by the ubiquitin-proteasome pathway, are requiredfor signal induced activation of NF-κB.

The observation that ubiquitin-mediated proteasomal proteolysis plays acritical role in the activation of NF-κB could be exploited clinicallyby the use of inhibitors directed toward the proteasome. Abnormalactivation of NF-κB followed by the stimulation of cytokine synthesishas been observed in a variety of inflammatory and infectious diseases.Activation of NF-κB is also essential for angiogenesis and forexpression of adhesion molecules (CAMs and selects), thus proteasomeinhibitors may also have utility in the treatment of diseases associatedwith the vascular system.

It is well documented that the ubiquitin-proteasome pathway is criticalfor the regulated destruction of cyclins that govern the exit frommitosis and allow cells to progress into the next phase of the cellcycle (Glotzer, M., Murray, A. W. and Kirschner, M. W. (1991) Nature349, 132-138). Thus, inhibiting the degradation of cyclins by usingproteasome inhibitors causes growth arrest. Therefore another potentialutility of proteasome inhibitors is their use in the treatment ofdiseases that result from abberrant cell division.

Several classes of peptidic inhibitors of 20S proteasome have beenreported in the recent literature. The α-ketoamide group has been usedin protease inhibitors for numerous indications. Specifically, a seriesof α-ketocarbonyl and boronic ester derived dipeptides (Iqbal, M.,Chatterjee, S., Kauer, J. C., Mallamo, J. P., Messina, P. A., Reiboldt,A. and Siman, R. 1996 Bioorg. Med. Chem. Lett 6, 287-290) have beenreported as potent inhibitors of 20S proteasomal function. Derivativesof 3-indolepyruvic acid have been claimed as pharmaceutically activecompounds for the treatment of disturbances of the central nervoussystem (De Luca, et al WO 88/09789) through a mechanism that modulateskynurenic acid levels in the brain.

Even though various compositions have been discovered that inhibit cellproliferation to some degree, there remains a need for more potentcompounds that inhibit cell proliferation via the 20S proteasome.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a method for inhibitingcell proliferation in mammals that uses a therapeutically effectiveamount of a composition heretofore unknown for its cell proliferativeinhibition properties.

It is also an object of this invention to provide a method for thetreatment of diseases that may be affected by the inhibition ofproteosomal function.

Further, it is an object of this invention to provide a method for thetreatment of proliferative diseases that operates by inhibitingproteasomal function.

It is another object of this invention to use a therapeuticallyeffective amount of the compositions described herein to inhibit cellproliferative disorders in humans.

Yet another object of this invention is the use of a therapeuticallyeffective amount of the compositions described herein to inhibitproteasomal function.

In one embodiment, this invention is a composition of matter having theformula: ##STR2## wherein X₂ is Ar or Ar--X₃ wherein X₃ is --C═O, or--CH₂ CO--, and wherein Ar is phenyl, substituted phenyl, indole,substituted indoles, and any other heteroaryls; R₁, and R₂ are eachindividually selected from the side chains of the known natural α-aminoacids and unnatural amino acids, hydrogen, 1-10 carbon linear andbranched alkyl, 1-10 carbon linear and branched substituted alkyl, aryl,substituted aryl, 1-10 carbon linear, branched substituted aryl,alkoxyaryl, 3-8 carbon cycloalkyl, heterocycle substituted heterocycle,heteroaryl and substituted heteroaryl; X₁ is selected from hydroxide,monoalkylamino, dialkylamino, alkoxide, arylkoxide and ##STR3## whereinX₄ is hydroxide, arylamino, monoalkylamino, dialkylamino, alkoxide, orarylalkoxide; and

R₃ is selected from the known natural α-amino acids, unnatural aminoacids, hydrogen, 1-10 carbon linear and branched alkyl, 1-10 carbonlinear and branched substituted alkyl, aryl, substituted aryl, 1-10carbon linear and branched substituted aryl, alkoxyaryl, 3-8 carboncycloalkyl, heterocycle, substituted heterocycle, heteroaryl andsubstituted heteroaryl.

In another embodiment, this invention is a method for inhibitingproteasomal protease factor in mammals comprising administering atherapeutically effective amount of the composition disclosed above tothe mammal.

In still another embodiment, this invention is a pharmaceuticalcomposition of matter comprising the composition of claim 1 and one ormore pharmaceutical excipients.

DESCRIPTION OF THE CURRENT EMBODIMENT

The invention is a method for inhibiting cell proliferation disorders,infectious diseases, and immunological diseases in mammals, andespecially in humans, using compositions having the following generalformula: ##STR4## where: X₂ is Ar or Ar--X₃ wherein X₃ is --C═O, --CH₂CO--, or (CH₂)n where n=0-2 and wherein

Ar is phenyl, substituted phenyl, indole, substituted indoles, and anyother heteroaryl.

R₁, and R₂ are each individually selected from the side chains of theknown natural (α-amino acids and unnatural amino acids: hydrogen, 1-10carbon linear and branched alkyl, 1-10 carbon linear and branchedsubstituted alkyl, aryl and substituted aryl, 1-10 carbon linear andbranched substituted aryl, alkoxyaryl, 3-8 carbon cycloalkyl,heterocycle and substituted heterocycle, or heteroaryl and substitutedheteroaryl. R₂ is preferably biaryl or biphenyl. R₁ is preferablyisobutyl. X₁ is selected from --OH, mono or dialkylamino, alkoxide,arylkoxide and ##STR5## wherein: X₄ is --OH, arylamino, mono ordialkylamino, alkoxide, or arylalkoxide; and preferably --OH

R₃ is selected from the side chains of known natural α-amino acids andunnatural amino acids, hydrogen, 1-10 carbon linear alkyl and branchedalkyl substituents, 1-10 carbon linear and branched substituted alkyl,aryl and substituted aryl, 1-10 carbon linear and branched substitutedaryl, alkoxyaryl, 3-8 carbon cycloalkyl, heterocycle and substitutedheterocycle, or heteroaryl and substituted heteroaryl. R3 is preferablyCO₂ H, CH₂ CO₂ H, (CH₂)₂ CO₂ H, Arg, Lys, Asn, Gln, Asp, Glu, Phe, andNle.

The following are definitions for certain terms used herein.

"Halogen" refers to fluorine, bromine, chlorine, and iodine atoms.

"Hydroxyl" refers to the group --OH.

"Thiol" or "mercapto" refers to the group --SH.

"alkyl" refers to a cyclic, branched or straight chain, alkyl group ofone to ten carbon atoms. This term is further exemplified by such groupsas methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl (or3-methylpropyl), cyclopropylmethyl, i-amyl, n-amyl, n-hexyl and thelike.

"Substituted alkyl" refers to lower alkyl as just described includingone or more groups such as hydroxyl, thiol, alkylthiol, halogen, alkoxy,amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, heterocycle,cycloheteroalkyl, substituted cycloheteroalkyl, acyl, carboxyl, aryl,substituted aryl, aryloxy, hetaryl, substituted hetaryl, aralkyl,heteroaralkyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkylcycloheteroalkyl, cyano. These groups may be attached to any carbon atomof the lower alkyl moiety.

"Aryloxy" denotes groups --OAr, where Ar is an aryl, substituted aryl,heteroaryl, or substituted heteroaryl group as defined below.

"Amino" denotes the group NRR', where R and R' may independently behydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl,hetaryl, or substituted hetaryl as defined below or acyl.

"Amido" denotes the group --C(O)NRR', where R and R' may independentlyby hydrogen, lower alkyl, substituted lower alkyl, aryl, substitutedaryl, hetaryl, substituted hetaryl as defined below.

"Carboxyl" denotes the group --C(O)OR, where R may independently behydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl,hetaryl, substituted hetaryl and the like as defined.

"Aryl" or "Ar" refers to an aromatic carbocyclic group having at leastone aromatic ring (e.g., phenyl or biphenyl) or multiple condensed ringsin which at least one ring is aromatic, (e.g.,1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl).

"Substituted aryl" refers to aryl optionally substituted with one ormore functional groups, e.g., halogen, lower alkyl, lower alkoxy,alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy,heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol,sulfamido and the like.

"Heterocycle" refers to a saturated, unsaturated, or aromaticcarbocyclic group having a single ring (e.g., morpholino, pyridyl orfuryl) or multiple condensed rings (e.g., naphthpyridyl, quinoxalyl,quinolinyl, indolizinyl or benzo[b]thienyl) and having at least onehetero atom, such as N, O or S, within the ring, which can optionally beunsubstituted or substituted with, e.g., halogen, lower alkyl, loweralkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl,aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol,sulfamido and the like.

"Heteroaryl" or "hetar" refers to a heterocycle in which at least oneheterocyclic ring is aromatic. Preferred heteroaryls are phenyl,substituted phenyl, indole and substituted indoles.

"Substituted heteroaryl" refers to a heterocycle optionally mono or polysubstituted with one or more functional groups, e.g., halogen, loweralkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl,hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl,nitro, cyano, thiol, sulfamido and the like.

"Cycloalkyl" refers to a divalent cyclic or polycyclic alkyl groupcontaining 3 to 15 carbon atoms.

"Substituted cycloalkyl" refers to a cycloalkyl group comprising one ormore substituents with, e.g., halogen, lower alkyl, substituted loweralkyl, alkoxy, alkylthio, acetylene, aryl, aryloxy, heterocycle,hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and thelike.

Examples of compounds that may be useful in the therapeutic methods ofthis invention, and specifically, useful as inhibitors of proteosomalfunction, are identified in Table 1 below:

Examples of compounds that may be useful in the therapeutic method ofthis invention (specifically, useful as inhibitors of proteosomalfunction) are listed in the table below:

                                      TABLE I                                     __________________________________________________________________________    Compositions used to inhibit 20S proteosome                                   #  Ar  X.sub.3                                                                           R.sub.2         R.sub.1 X.sub.1                                                                            R.sub.3         X.sub.4               __________________________________________________________________________      1 phenyl CH.sub.2 CO                                                                                                                  #STR6##                                                                       OH R7##                - 2 Indole CH.sub.2 CO                                                                                                               #STR8##                                                                       OH R9##                - 3 Indole CH.sub.2 CO                                                                                                               #STR10##                                                                      PhCH.sub.2 N                                                                  - 4 Indole  CO                                                                #STR12##                                                                       STR13##                                                                      OH R14##               - 5 Indole CH.sub.2 CO                                                                                                               #STR15##                                                                       STR16##                                                                      OH R17##               - 6 phenyl CH.sub.2 CO                                                                                                               #STR18##                                                                       STR19##                                                                      OH R20##               - 7 phenyl CH.sub.2 CO                                                                                                               #STR21##                                                                      #STR22##                                                                      PhCH.sub.2 N                                                                  - 8 Indole CO                                                                 #STR24##                                                                       STR25##                                                                      PhCH.sub.2 N                                                                  - 9 Indole                                                                  CH.sub.2 CO                                                                     #STR27##                                                                       STR28##                                                                      PhCH.sub.2 N                                                                  - 10 Indole CO                                                                #STR30##                                                                      OH R31##               - 11 phenyl CO                                                                                                                       #STR32##                                                                      OH R33##               - 12 phenyl CO                                                                                                                       #STR34##                                                                       STR35##                                                                      OH R36##               - 13 phenyl CH.sub.2 CO                                                                                                              #STR37##                                                                       STR38##                                                                      OH R39##               - 14 Indole CO                                                                                                                       #STR40##                                                                       STR41##                                                                      OH R42##               - 15 Indole CH.sub.2 CO                                                                                                              #STR43##                                                                       STR44##                                                                      OH R45##               - 16 phenyl CO                                                                                                                       #STR46##                                                                       STR47##                                                                      OH R48##               - 17 Indole CH.sub.2 CO                                                                                                              #STR49##                                                                       STR50##                                                                      OH R51##               - 18 Indole CH.sub.2 CO                                                                                                              #STR52##                                                                       STR53##                                                                      OH R54##               - 19 Indole CH.sub.2 CO                                                                                                              #STR55##                                                                       STR56##                                                                      OH R57##               - 20 Indole CH.sub.2 CO                                                                                                              #STR58##                                                                       STR59##                                                                      OH R60##               - 21 Indole CH.sub.2 CO                                                                                                              #STR61##                                                                       STR62##                                                                      OH R63##               - 22 Indole CH.sub.2 CO                                                                                                              #STR64##                                                                       STR65##                                                                      OH R66##               - 23 Indole CH.sub.2 CO                                                                                                              #STR67##                                                                       STR68##                                                                      OH R69##               - 24 Indole CH.sub.2 CO                                                                                                              #STR70##                                                                       STR71##                                                                      OH R72##               - 25 Indole CH.sub.2 CO                                                                                                              #STR73##                                                                       STR74##                                                                      OH R75##               - 26 Indole CH.sub.2 CO                                                                                                              #STR76##                                                                       STR77##                                                                      OH R78##               - 27 Indole CH.sub.2 CO                                                                                                              #STR79##                                                                       STR80##                                                                      OH R81##               - 28 Indole CH.sub.2 CO                                                                                                              #STR82##                                                                       STR83##                                                                      OH R84##               - 29 Indole CH.sub.2 CO                                                                                                              #STR85##                                                                       STR86##                                                                      OH R87##               - 30 Indole CH.sub.2 CO                                                                                                              #STR88##                                                                       STR89##                                                                      OH R90##               - 31 Indole CH.sub.2 CO                                                                                                              #STR91##                                                                       STR92##                                                                      OH R93##               - 32 Indole CH.sub.2 CO                                                                                                              #STR94##                                                                       STR95##                                                                      OH R96##               - 33 Indole CH.sub.2 CO                                                                                                              #STR97##                                                                       STR98##                                                                      OH R99##               - 34 Indole CH.sub.2 CO                                                                                                              #STR100##                                                                      STR101##                                                                     OH R102##              - 35 Indole CH.sub.2 CO                                                                                                              #STR103##                                                                      STR104##                                                                     OH R105##              - 36 Indole CH.sub.2 CO                                                                                                              #STR106##                                                                      STR107##                                                                     OH R108##              - 37 Indole CH.sub.2 CO                                                                                                              #STR109##                                                                      STR110##                                                                     OH R111##              - 38 Indole CH.sub.2 CO β-Ala                                                                                                    STR112##                                                                     OH R113##              - 39 Indole CH.sub.2 CO                                                                                                              #STR114##                                                                      STR115##                                                                     OH R116##              - 40 Indole CH.sub.2 CO                                                                                                              #STR117##                                                                      STR118##                                                                     OH R119##              - 41 Indole CH.sub.2 CO                                                                                                              #STR120##                                                                      STR121##                                                                     OH R122##              - 42 Indole CH.sub.2 CO                                                                                                              #STR123##                                                                      STR124##                                                                     OH R125##              - 43 Indole CH.sub.2 CO                                                                                                              #STR126##                                                                      STR127##                                                                     OH R128##              - 44 Indole CH.sub.2 CO                                                                                                              #STR129##                                                                      STR130##                                                                     OH R131##              - 45 Indole CH.sub.2 CO                                                                                                              #STR132##                                                                      STR133##                                                                     OH R134##              - 46 Indole CH.sub.2 CO                                                                                                              #STR135##                                                                      STR136##                                                                     OH R137##              - 47 Indole CH.sub.2 CO                                                                                                              #STR138##                                                                      STR139##                                                                     OH R140##              - 48 Indole CH.sub.2 CO                                                                                                              #STR141##                                                                      STR142##                                                                     OH R143##              - 49 Indole CH.sub.2 CO                                                                                                              #STR144##                                                                      STR145##                                                                     OH R146##              - 50 Indole CH.sub.2 CO                                                                                                              #STR147##                                                                      STR148##                                                                     OH R149##              - 51 Indole CH.sub.2 CO                                                                                                              #STR150##                                                                      STR151##                                                                     OH R152##              - 52 Indole CH.sub.2 CO                                                                                                              #STR153##                                                                      STR154##                                                                     OH R155##              - 53 Indole CH.sub.2 CO β-Ala                                                                                                    STR156##                                                                     OH R157##              - 54 Indole CH.sub.2 CO                                                                                                              #STR158##                                                                      STR159##                                                                     OH R160##              - 55 Indole CH.sub.2 CO                                                                                                              #STR161##                                                                      STR162##                                                                     OH R163##              - 56 Indole CH.sub.2 CO                                                                                                              #STR164##                                                                      STR165##                                                                     OH R166##              - 57 Indole CH.sub.2 CO                                                                                                              #STR167##                                                                      STR168##                                                                     OH R169##              - 58 Indole CH.sub.2 CO                                                                                                              #STR170##                                                                      STR171##                                                                     OH R172##              - 59 Indole CH.sub.2 CO                                                                                                              #STR173##                                                                      STR174##                                                                     OH R175##              - 60 Indole CH.sub.2 CO                                                                                                              #STR176##                                                                      STR177##                                                                     OH R178##              - 61 Indole CH.sub.2 CO                                                                                                              #STR179##                                                                      STR180##                                                                     OH R181##              - 62 Indole CH.sub.2 CO                                                                                                              #STR182##                                                                      STR183##                                                                     OH R184##              - 63 Indole CH.sub.2 CO                                                                                                              #STR185##                                                                      STR186##                                                                     OH R187##              - 64 Indole CH.sub.2 CO                                                                                                              #STR188##                                                                      STR189##                                                                     OH R190##              - 65 Indole CH.sub.2 CO                                                                                                              #STR191##                                                                      STR192##                                                                     OH R193##              - 66 Indole CH.sub.2 CO                                                                                                              #STR194##                                                                      STR195##                                                                     OH R196##              - 67 Indole CH.sub.2 CO                                                                                                              #STR197##                                                                      STR198##                                                                     OH R199##              - 68 Indole CH.sub.2 CO β-Ala                                                                                                    STR200##                                                                     OH R201##              - 69 Indole CH.sub.2 CO                                                                                                              #STR202##                                                                      STR203##                                                                     OH R204##              - 70 Indole CH.sub.2 CO                                                                                                              #STR205##                                                                      STR206##                                                                     OH R207##              - 71 Indole CH.sub.2 CO                                                                                                              #STR208##                                                                      STR209##                                                                     OH R210##              - 72 Indole CH.sub.2 CO                                                                                                              #STR211##                                                                      STR212##                                                                     OH R213##              - 73 Indole CH.sub.2 CO                                                                                                              #STR214##                                                                      STR215##                                                                     OH R216##              - 74 Indole CH.sub.2 CO                                                                                                              #STR217##                                                                      STR218##                                                                     OH R219##              - 75 Indole CH.sub.2 CO                                                                                                              #STR220##                                                                      STR221##                                                                     OH R222##              - 76 Indole CH.sub.2 CO                                                                                                              #STR223##                                                                      STR224##                                                                     OH R225##              - 77 Indole CH.sub.2 CO                                                                                                              #STR226##                                                                      STR227##                                                                     OH R228##              - 78 Indole CH.sub.2 CO                                                                                                              #STR229##                                                                      STR230##                                                                     OH R231##              - 79 Indole CH.sub.2 CO                                                                                                              #STR232##                                                                      STR233##                                                                     OH R234##              - 80 Indole CH.sub.2 CO                                                                                                              #STR235##                                                                      STR236##                                                                     OH R237##              - 81 Indole CH.sub.2 CO                                                                                                              #STR238##                                                                      STR239##                                                                     OH R240##              - 82 Indole CH.sub.2 CO                                                                                                              #STR241##                                                                      STR242##                                                                     OH R243##              - 83 Indole CH.sub.2 CO                                                                                                              #STR244##                                                                      STR245##                                                                     OH R246##              - 84 Indole CH.sub.2 CO β-Ala                                                                                                    STR247##                                                                     OH R248##              - 85 Indole           CO                                                                                                             #STR249##                                                                      STR250##                                                                     OH R251##              - 86 Indole CH.sub.2 CO                                                                                                              #STR252##                                                                      STR253##                                                                     OH R254##              - 87 Indole CH.sub.2 CO                                                                                                              #STR255##                                                                      STR256##                                                                     OH R257##              - 88 Indole CH.sub.2 CO                                                                                                              #STR258##                                                                      STR259##                                                                     OH R260##              - 89 Indole CH.sub.2 CO                                                                                                              #STR261##                                                                      STR262##                                                                     OH R263##              - 90 Indole CH.sub.2 CO β-Ala                                                                                                    STR264##                                                                     OH R265##              - 91 Indole CH.sub.2 CO                                                                                                              #STR266##                                                                      STR267##                                                                     OH R268##              - 92 Indole CH.sub.2 CO                                                                                                              #STR269##                                                                      STR270##                                                                     OH R271##              - 93 Indole CH.sub.2 CO                                                                                                              #STR272##                                                                      STR273##                                                                     OH R274##              - 94 Indole CH.sub.2 CO                                                                                                              #STR275##                                                                      STR276##                                                                     OH R277##              - 95 Indole CH.sub.2 CO                                                                                                              #STR278##                                                                      STR279##                                                                     OH R280##              - 96 Indole CH.sub.2 CO                                                                                                              #STR281##                                                                      STR282##                                                                     OH R283##              - 97 Indole CH.sub.2 CO                                                                                                              #STR284##                                                                      STR285##                                                                     OH R286##              - 98 Indole CH.sub.2 CO                                                                                                              #STR287##                                                                      STR288##                                                                     OH R289##              - 99 Indole CH.sub.2 CO                                                                                                              #STR290##                                                                      STR291##                                                                     OH R292##              - 100 Indole CH.sub.2 CO                                                                                                             #STR293##                                                                      STR294##                                                                     OH R295##              - 101 Indole CH.sub.2 CO                                                                                                             #STR296##                                                                      STR297##                                                                     OH R298##              - 102 Indole CH.sub.2 CO                                                                                                             #STR299##                                                                      STR300##                                                                     OH R301##              - 103 Indole CH.sub.2 CO                                                                                                             #STR302##                                                                      STR303##                                                                     OH R304##              - 104 Indole CH.sub.2 CO                                                                                                             #STR305##                                                                      STR306##                                                                     OH R307##              - 105 Indole CH.sub.2 CO β-Ala                                                                                                   STR308##                                                                     OH R309##              - 106 Indole CH.sub.2 CO                                                                                                             #STR310##                                                                      STR311##                                                                     OH R312##              - 107 Indole CH.sub.2 CO                                                                                                             #STR313##                                                                      STR314##                                                                     OH R315##              - 108 Indole CH.sub.2 CO                                                                                                             #STR316##                                                                      STR317##                                                                     OH R318##              - 109 Indole CH.sub.2 CO                                                                                                             #STR319##                                                                      STR320##                                                                     OH R321##              - 110 Indole CH.sub.2 CO                                                                                                             #STR322##                                                                      STR323##                                                                     OH R324##              - 111 Indole CH.sub.2 CO Val                                                                                                          STR325##                                                                     OH R326##              - 112 Indole CH.sub.2 CO Nva                                                                                                          STR327##                                                                     OH R328##              - 113 Indole CH.sub.2 CO                                                                                                             #STR329##                                                                      STR330##                                                                     OH R331##              - 114 Indole CH.sub.2 CO                                                                                                             #STR332##                                                                      STR333##                                                                     OH R334##              - 115 Indole CH.sub.2 CO                                                                                                             #STR335##                                                                      STR336##                                                                     OH R337##              - 116 Indole CH.sub.2 CO                                                                                                             #STR338##                                                                      STR339##                                                                     OH R340##              - 117 Indole CH.sub.2 CO                                                                                                             #STR341##                                                                      STR342##                                                                     OH R343##              - 118 Indole CH.sub.2 CO                                                                                                             #STR344##                                                                      STR345##                                                                     OH R346##              - 119 Indole CH.sub.2 CO                                                                                                             #STR347##                                                                      STR348##                                                                     OH R349##              - 120 Indole CH.sub.2 CO β-Ala                                                                                                   STR350##                                                                     OH R351##              - 121 Indole CH.sub.2 CO                                                                                                               TR352##                                                                      STR353##                                                                     OH R354##              - 122 Indole CH.sub.2 CO                                                                                                               TR355##                                                                      STR356##                                                                     OH R357##              - 123 Indole CH.sub.2 CO                                                                                                               TR358##                                                                      STR359##                                                                     OH R360##              - 124 Indole CH.sub.2 CO                                                                                                               TR361##                                                                      STR362##                                                                     OH R363##              - 125 Indole CH.sub.2 CO                                                                                                               TR364##                                                                      STR365##                                                                     OH R366##              - 126 Indole CH.sub.2 CO                                                                                                               TR367##                                                                      STR368##                                                                     OH R369##              - 127 Indole CH.sub.2 CO                                                                                                               TR370##                                                                      STR371##                                                                     OH R372##              - 128 Indole CH.sub.2 CO                                                                                                               TR373##                                                                      STR374##                                                                     OH R375##              - 129 Indole CH.sub.2 CO                                                                                                               TR376##                                                                      STR377##                                                                     OH R378##              - 130 Indole CH.sub.2 CO                                                                                                               TR379##                                                                      STR380##                                                                     OH R381##              - 131 Indole CH.sub.2 CO                                                                                                               TR382##                                                                      STR383##                                                                     OH R384##              - 132 Indole CH.sub.2 CO                                                                                                               TR385##                                                                      STR386##                                                                     OH R387##              - 133 Indole CH.sub.2 CO                                                                                                               TR388##                                                                      STR389##                                                                     OH R390##              - 134 Indole CH.sub.2 CO                                                                                                               TR391##                                                                      STR392##                                                                     OH R393##              - 135 Indole CH.sub.2 CO β-Ala                                                                                                   STR394##                                                                     OH R395##              - 136 Indole CH.sub.2 CO                                                                                                               TR396##                                                                      STR397##                                                                     OH R398##              - 137 Indole CH.sub.2 CO                                                                                                               TR399##                                                                      STR400##                                                                     OH R401##              - 138 Indole CH.sub.2 CO                                                                                                               TR402##                                                                      STR403##                                                                     OH R404##              - 139 Indole CH.sub.2 CO                                                                                                               TR405##                                                                      STR406##                                                                     OH R407##              - 140 Indole CH.sub.2 CO                                                                                                               TR408##                                                                      STR409##                                                                     OH R410##              - 141 Indole CH.sub.2 CO                                                                                                               TR411##                                                                      STR412##                                                                     OH R413##              - 142 Indole CH.sub.2 CO                                                                                                               TR414##                                                                      STR415##                                                                     OH R416##              - 143 Indole CH.sub.2 CO                                                                                                               TR417##                                                                      STR418##                                                                     OH R419##              - 144 Indole CH.sub.2 CO                                                                                                               TR420##                                                                      STR421##                                                                     OH R422##              - 145 Indole CH.sub.2 CO                                                                                                               TR423##                                                                      STR424##                                                                     OH R425##              - 146 Indole CH.sub.2 CO                                                                                                               TR426##                                                                      STR427##                                                                     OH R428##              - 147 Indole CH.sub.2 CO                                                                                                               TR429##                                                                      STR430##                                                                     OH R431##              - 148 Indole CH.sub.2 CO 4,4'-BPA                                                                                                     STR432##                                                                     OH R433##              - 149 Indole CH.sub.2 CO                                                                                                               TR434##                                                                      STR435##                                                                     OH R436##              - 150 Indole CH.sub.2 CO β-Ala                                                                                                   STR437##                                                                     OH R438##              - 151 Indole CH.sub.2 CO                                                                                                               TR439##                                                                      STR440##                                                                     OH R441##              - 152 Indole CH.sub.2 CO                                                                                                               TR442##                                                                      STR443##                                                                     OH R444##              - 153 Indole CH.sub.2 CO                                                                                                               TR445##                                                                      STR446##                                                                     OH R447##              - 154 Indole CH.sub.2 CO                                                                                                               TR448##                                                                      STR449##                                                                     OH R450##              - 155 Indole CH.sub.2 CO                                                                                                               TR451##                                                                      STR452##                                                                     OH R453##              - 156 Indole CH.sub.2 CO                                                                                                               TR454##                                                                      STR455##                                                                     OH R456##              - 157 Indole CH.sub.2 CO                                                                                                               TR457##                                                                      STR458##                                                                     OH R459##              - 158 Indole CH.sub.2 CO                                                                                                               TR460##                                                                      STR461##                                                                     OH R462##              - 159 Indole CH.sub.2 CO                                                                                                               TR463##                                                                      STR464##                                                                     OH R465##              - 160 Indole CH.sub.2 CO                                                                                                               TR466##                                                                      STR467##                                                                     OH R468##              - 161 Indole CH.sub.2 CO                                                                                                               TR469##                                                                      STR470##                                                                     OH R471##              - 162 Indole CH.sub.2 CO                                                                                                               TR472##                                                                      STR473##                                                                     OH R474##              - 163 Indole CH.sub.2 CO                                                                                                               TR475##                                                                      STR476##                                                                     OH R477##              - 164 Indole CH.sub.2 CO                                                                                                               TR478##                                                                      STR479##                                                                     OH R480##              - 165 Indole CH.sub.2 CO                                                                                                               TR481##                                                                      STR482##                                                                     OH R483##              - 167 Indole CH.sub.2 CO                                                                                                               TR484##                                                                      STR485##                                                                     OH R486##              - 168 Indole CH.sub.2 CO                                                                                                               TR487##                                                                      STR488##                                                                     OH R489##              - 169 Indole CH.sub.2 CO                                                                                                               TR490##                                                                      STR491##                                                                     OH R492##              - 170 Indole CH.sub.2 CO                                                                                                               TR493##                                                                      STR494##                                                                     OH R495##              - 171 Indole CH.sub.2 CO                                                                                                               TR496##                                                                      STR497##                                                                     OH R498##              - 172 Indole CH.sub.2 CO                                                                                                               TR499##                                                                      STR500##                                                                     OH R501##              - 173 Indole CH.sub.2 CO                                                                                                               TR502##                                                                      STR503##                                                                     OH R504##              - 174 Indole CH.sub.2 CO                                                                                                               TR505##                                                                      STR506##                                                                     OH R507##              - 175 Indole CH.sub.2 CO                                                                                                               TR508##                                                                      STR509##                                                                     OH R510##              - 176 Indole CH.sub.2 CO                                                                                                               TR511##                                                                      STR512##                                                                     OH R513##              - 177 Indole CH.sub.2 CO                                                                                                               TR514##                                                                      STR515##                                                                     OH R516##              - 178 Indole CH.sub.2 CO                                                                                                               TR517##                                                                      STR518##                                                                     OH R519##              - 179 Indole CH.sub.2 CO                                                                                                               TR520##                                                                      STR521##                                                                     OH R522##              - 180 Indole CH.sub.2 CO                                                                                                               TR523##                                                                      STR524##                                                                     OH R525##              - 181 Indole CH.sub.2 CO                                                                                                               TR526##                                                                      STR527##                                                                     OH R528##              - 182 Indole CH.sub.2 CO                                                                                                               TR529##                                                                      OH 530##              - 183 Indole CH.sub.2 CO                                                                                                               TR531##                                                                      STR532##                                                                     OH R533##              - 184 Indole CH.sub.2 CO                                                                                                               TR534##                                                                      STR535##                                                                     OH R536##              - 185 Indole CH.sub.2 CO                                                                                                               TR537##                                                                      STR538##                                                                     OH R539##              - 186 Indole CH.sub.2 CO                                                                                                               TR540##                                                                      STR541##                                                                     OH R542##              - 187 Indole CH.sub.2 CO                                                                                                               TR543##                                                                      STR544##                                                                     OH R545##              - 188 Indole CH.sub.2 CO                                                                                                               TR546##                                                                      β-Alanine                                                             OH                       - 189 Indole CH.sub.2 CO                                                                                                               TR548##                                                                      STR549##                                                                     OH R550##              - 190 Indole CH.sub.2 CO                                                                                                               TR551##                                                                      STR552##                                                                     OH R553##              - 191 Indole CH.sub.2 CO                                                                                                               TR554##                                                                      STR555##                                                                     OH R556##              - 192 Indole CH.sub.2 CO                                                                                                               TR557##                                                                      STR558##                                                                     OH R559##              - 193 Indole CH.sub.2 CO                                                                                                               TR560##                                                                      STR561##                                                                     OH R562##              - 194 Indole CH.sub.2 CO                                                                                                               TR563##                                                                      STR564##                                                                     OH R565##              - 195 Indole CH.sub.2 CO                                                                                                               TR566##                                                                      STR567##                                                                     OH R568##              - 196 Indole CH.sub.2 CO                                                                                                               TR569##                                                                      STR570##                                                                     OH R571##              - 197 Indole CH.sub.2 CO                                                                                                               TR572##                                                                      STR573##                                                                     OH R574##              - 198 Indole CH.sub.2 CO                                                                                                               TR575##                                                                      STR576##                                                                     OH R577##              - 199 Indole CH.sub.2 CO                                                                                                               TR578##                                                                      STR579##                                                                     OH R580##              - 200 Indole CH.sub.2 CO                                                                                                               TR581##                                                                      STR582##                                                                     OH R583##              - 201 Indole CH.sub.2 CO                                                                                                               TR584##                                                                      STR585##                                                                     OH R586##              - 202 Indole CH.sub.2 CO                                                                                                               TR587##                                                                      STR588##                                                                     OH R589##              - 203 Indole CH.sub.2 CO                                                                                                               TR590##                                                                      STR591##                                                                     OH R592##              - 204 Indole CH.sub.2 CO                                                                                                               TR593##                                                                      STR594##                                                                     OH R595##              - 205 Indole CH.sub.2 CO                                                                                                               TR596##                                                                      STR597##                                                                     OH R598##              - 206 Indole CH.sub.2 CO                                                                                                               TR599##                                                                      STR600##                                                                     OH R601##              - 207 Indole CH.sub.2 CO β-Alanine   β-Alanine   OH              __________________________________________________________________________

The compounds described above are useful for treating diseases anddisorders mediated by the 20S proteasome such as antiproliferativediseases, cancer, inflammation. It is preferred that the compositions ofthis invention are used to treat antiproliferative disorders andinflammation. It is most preferred that the compounds of this inventionare used to treat inflammatory diseases.

The compounds of the present invention are useful for treating disordersmediated by 20S proteasome in mammals.

The compounds of this invention may be administered to mammals bothprophylactically and therapeutically by any administration protocol thatis capable of supplying at least one compound of this invention to a 20Sproteasome. Non-limiting examples of useful administration protocolsinclude orally, parenterally, dermally, transdermally, rectally, nasallyor by any other suitable pharmaceutical composition administrationprotocol that is within the knowledge of one skilled in the art.

The compositions of this invention may be administered in suitablepharmaceutical dosage forms. The pharmaceutical dosage form will dependlargely upon the administration protocol used. The term pharmaceuticaldosage form refers to items such as tablets, capsules, liquids andpowders, comprising 20S proteasome inhibitors of this invention alone orin the presence of one or more pharmaceutical excipients. The choice ofadditives such as excipients and adjuvants again will depend largelyupon the chosen administration protocol. Those skilled in thepharmaceutical arts will recognize a wide variety of formulations andvehicles for administering compositions of this invention.

The administration protocol chosen for compounds of this invention willultimately dictate the final form and composition of pharmaceuticaldosage forms comprising the 20S proteasome inhibitors of this invention.For example, internal administration of compounds of this invention iseffected, orally, in the form of powders, tablets, capsules, pastes,drinks, granules, or solutions, suspensions and emulsions which can beadministered orally, or bolus, in medicated food, or in drinking water.Internal administration may also be accomplished using a timed releaseformulation including additives such as surfactant or starch coatedcapsules, or using a quick release formulation such as a freeze-driedfast dissolving tablet. Dermal administration is effected, for example,in the form of transdermal patches, spraying or pouring-on andspotting-on. Parenteral administration is effected, for example, in theform of injection (intramuscularly, subcutaneously, intravenously,intraperitoneally) or by implants.

Suitable pharmaceutical dosage forms incorporating the 20S proteasomeinhibitors of this invention include but are not limited to solutionssuch as solutions for injection, oral solutions, concentrates for oraladministration after dilution, solutions for use on the skin or in bodycavities, pour-on and spot-on formulations, gels; emulsions andsuspension for oral or dermal administration and for injection;semi-solid preparations; formulations in which the active compound isincorporated in cream base or in an oil-in-water or water-in-oilemulsion base; solid preparations such as powders, premixes orconcentrates, granules, pellets, tablets, boli, capsules; aerosols andinhalants, and shaped articles containing active compound.

Pharmaceutical dosage forms that are solutions may be administered byinjection intravenously, intramuscularly and subcutaneously. Solutionsfor injection are prepared by dissolving the active compound in asuitable solvent and, if appropriate, adding adjuvants such assolubilizers, acids, bases, buffer salts, antioxidants andpreservatives. The solutions are sterile-filtered and drawn off.

Alternatively, solutions including compositions of this invention may beadministered orally. Concentrates of compositions of this invention arepreferably administered orally only after diluting the concentrate tothe administration concentration. Oral solutions and concentrates areprepared as described above in the case of the solutions for injection.Solutions for use on the skin are applied dropwise, brushed on, rubbedin, splashed on or sprayed on. These solutions are prepared as describedabove in the case of solutions for injection.

Gels are applied to the skin, or introduced into body cavities. Gels areprepared by treating solutions which have been prepared as described inthe case of the solutions for injection with such an amount of thickenerthat a clear substance of cream-like consistency is formed, or by anyother means known to one skilled in the art.

Pour-on and spot-on formulations are poured onto, or splashed onto,limited areas of the skin, the active compound penetrating the skin andacting systemically. Pour-on and spot-on formulations are prepared bydissolving, suspending or emulsifying the active compound in suitablesolvents or solvent mixtures which are tolerated by the skin. Ifappropriate, other adjuvants such as colorants, resorption accelerators,antioxidants, light stabilizers, and tackifiers are added.

Emulsions can be administered orally, dermally or in the form ofinjections. Emulsions are either of the water-in-oil type or of theoil-in-water type. They are prepared by dissolving 20S proteasomeinhibitors either in the hydrophobic or in the hydrophilic phase andhomogenizing the phase with a solvent of the opposite phase with the aidof suitable adjuvants such as emulsifiers, colorants, resorptionaccelerators, preservatives, antioxidants, light stabilizers, andviscosity-increasing substances.

Suspensions can be administered orally, dermally or in the form ofinjection. They are prepared by suspending the active compound in aliquid if appropriate with the addition of further adjuvants such aswetting agents, colorants, resorption accelerators, preservatives,antioxidants and light stabilizers.

The pharmaceutical compositions of this invention may include one ormore additives in the form of pharmaceutically acceptable additives.Useful additives include solvents, solubilizers, preservatives,thickeners, wetting agents, colorants, resorption accelerators,antioxidants, light stabilizers, tackifiers, viscosity increasingsubstances, fillers, flavorings, lubricating agents, and any otherpharmaceutical composition additive known to those skilled in the art.

The additive may be a solvent such as water, alcohols such as ethanol,butanol, benzyl alcohol, glycerol, propylene glycol, polyethyleneglycols, N-methyl-pyrrolidone, alkanols, glycerol, aromatic alcoholssuch as benzyl alcohol, phenylethanol, phenoxyethanol, esters such asethyl acetate, butyl acetate, benzyl benzoate, ethers such as alkyleneglycol alkyl ethers such as dipropylene glycol mono-methyl ether,diethylene glycol mono-butyl ether, ketones such as acetone, methylethyl ketone, aromatic and/or aliphatic hydrocarbons, vegetable orsynthetic oils, DMF, dimethylacetamide, N-methyl-pyrrolidone,2,2-dimethyl-4-oxy-methylene-1,3-dioxolane.

The following additives may be useful as solubilizers of thecompositions of this invention: solvents which enhance solution of theactive compound in the main solvent or which prevent its precipitation.Examples are polyvinylpyrrolidone, polyoxyethylated castor oil,polyoxyethylated sorbitan esters.

Useful preservatives are, for example, benzyl alcohol, trichlorobutanol,p-hydroxybenzoic esters, and n-butanol.

Useful thickeners include inorganic thickeners such as bentonite,colloidal silica, aluminum monostearate, organic thickeners such ascellulose derivatives, polyvinyl alcohols and their copolymers,acrylates and methacrylates.

Other liquids which may be useful in pharmaceutical dosage forms of thisinvention are, for example, homogeneous solvents, solvent mixtures, andwetting agents which are typically surfactants.

Useful colorants are all colorants which are non-toxic and which can bedissolved or suspended.

Useful resorption accelerators are DMSO, spreading oils such asisopropyl myristate, dipropylene glycol pelargonate, silicone oils,fatty acid esters, triglycerides, fatty alcohols.

Useful antioxidants are sulphites or metabisulphites such as potassiummetabisulphite, ascorbic acid, butylhydroxytoluene, butylhydroxyanisole,tocopherol.

A useful light stabilizer is novantisolic acid.

Useful tackifiers include cellulose derivatives, starch derivatives,polyacrylates, natural polymers such as alginates, gelatin.

Useful emulsifiers include non-ionic surfactants such aspolyoxyethylated castor oil, polyoxyethylated sorbitan monooleate,sorbitan monostearate, glycerol monostearate, polyoxyethyl stearate,alkylphenol polyglycol ethers; ampholytic surfactants such as Di-NaN-lauryl-beta-iminodipropionate or lecithin; anionic surfactants, suchas Na-lauryl sulphate, fatty alcohol ether sulphates, themonoethanolamine salt of mono/dialkylpolyglycol ether orthophosphoricesters; cationic surfactants such as cetyltrimethylammonium chloride.

Useful viscosity-increasing substances and substances which stabilize atherapeutic emulsion include carboxymethylcellulose, methylcellulose andother cellulose and starch derivatives, polyacrylates, alginates,gelatin, gum Arabic, polyvinylpyrrolidone, polyvinyl alcohol, copolymersof methyl vinyl ether and maleic anhydride, polyethylene glycols, waxes,colloidal silica or mixtures of the substances mentioned.

To prepare solid pharmaceutical dosage forms, the active compound ismixed with suitable additives, if appropriate with the addition ofadjuvants, and the mixture is formulated as desired. Examples ofphysiologically acceptable solid inert additives include sodiumchloride, carbonates such as calcium carbonate, hydrogen carbonates,aluminum oxides, silicas, clays, precipitated or colloidal silicondioxide, and phosphates. Examples of solid organic additives includesugars, cellulose, foods such as dried milk, animal meals, cereal mealsand coarse cereal meals and starches. Other suitable additives includelubricants and gliding agents such as magnesium stearate, stearic acid,talc, bentonites; disintegrants such as starch or crosslinkedpolyvinylpyrrolidone; binders such as, starch, gelatin or linearpolyvinylpyrrolidone; and dry binders such as microcrystallinecellulose.

In the pharmaceutical dosage forms described herein, the activecompounds can be present in the form of a mixture with at least oneother 20S proteasome inhibitor. Alternatively, or in addition, thepharmaceutical dosage forms of the invention can, in addition to atleast one 20S proteasome inhibitor, include any pharmaceutical compoundthat is capable of treating any known malady or disorder where theadministration of both together create no unacceptable adverse effects.

Methods for treating 20S proteasome mediated diseases and disorderscomprises the administration of an effective quantity of the chosencompound or combinations thereof, preferably dispersed in apharmaceutical dosage form. Ready-to-use pharmaceutical dosage forms ofthis invention contain the active compound in concentrations of from 10ppm to 20 percent by weight, and preferably of from 0.1 to 10 percent byweight. Pharmaceutical dosage forms of this invention that are dilutedprior to administration, preferably contain the active compound inconcentrations of from 0.5 to 90 percent by weight, and preferably offrom 5 to 50 percent by weight. In general, it has proved advantageousto administer amounts of approximately 0.01 mg to approximately 100 mgof active compound per kg of body weight per day to achieve effectiveresults.

The amount and frequency of administration of pharmaceutical dosageforms comprising 20S proteasome inhibitors of this invention will bereadily determined by one skilled in the art depending upon, among otherfactors, the route of administration, age and condition of the patient.These dosage units may be administered one to ten times daily for acuteor chronic disease. No unacceptable toxicological effects are expectedwhen compounds of the invention are administered in accordance with thepresent invention.

The pharmaceutical dosage forms comprising 20S proteasome inhibitors ofthis invention are made following the conventional techniques ofpharmacy involving milling, mixing, granulation, and compressing, whennecessary, for tablet forms; or milling, mixing and filling for hardgelatin capsule forms. When a liquid additive is used, the preparationwill be in the form of a syrup, elixir, emulsion or an aqueous ornon-aqueous suspension. Such a liquid formulation may be administereddirectly p.o. or filled into a soft gelatin capsule.

While the compositions described herein may be administered as describedabove, it is preferred that the method of this invention is achieved byadministering the compound described herein orally. When the oraladministration route is chosen, a larger quantity of reactive agent willbe required to produce the same effect as a smaller quantity given forexample parenterally. In accordance with good clinical practice, it ispreferred to administer the compound according to this method at aconcentration level that would produce effective therapeutic resultswithout causing any harmful side effects.

The compositions of this invention have non-therapeutic utility as well.The compositions of this invention are useful as analytical standardsfor 20S proteasome inhibitor assays.

EXAMPLE 1

The compounds useful in the therapeutic method of this invention areprepared by conventional methods of organic chemistry. References thatmay be consulted in describing the art of the synthesis of thesecompounds include Bodansky's "The Practice of Peptide Synthesis,"Springer-Verlag, First Edition, 1984; "Protective Groups in OrganicSynthesis," Second Edition, John Wiley and Sons, New York, 1991. Allpeptide couplings are accomplished at room temperature with gentle andconstant agitation. Peptide couplings and deprotections are monitoredusing the Kaiser test for amines. Xaa refers to any of the commericallyavailable amino acids that may be purchased pre-attached to the MBHAresin. Yaa and Zaa refer to any of the commerically available aminoacids.

The compounds of this invention may be prepared by solid phase peptidesynthesis (SPPS) in the general procedure that follows: Xaa-MBHA-resinis weighed and transferred to a syringe equipped with a fritted filter.The resin is pre-swollen in DMF and then the N-terminal protecting groupis removed by treatment with 30%, piperidine in DMF for 30 minutes. Thedeprotection solution is removed. The deprotected resin is washed fivetimes with DMF, five times with MeOH, and then five times with DMF.Amino acid Yaa may then be coupled to the deprotected resin using asolution of Yaa in DMF containing 3 equivalents each of Yaa,carbodiimide coupling reagent and HOBT (hydroxy benzotriazole).Succesive couplings with solutions of Yaa may be necessary to achievecoupling efficiencies that pass the Kaiser test. The N-terminal groupdeprotection and Yaa coupling step may be repeated to couple a thirdamino acid Zaa. The final coupling step uses ketoacid, carbodiimide, andHOBT in DMF, and this step is repeated until the coupling passes theKaiser test. The completed peptide sequence on resin is dried undervacuum for at least six hours and then cleaved by treatment for 2.5hours with either 95/5 trifluoroacetic acid/water or a freshly preparedsolution of 90%, trifluoroacetic acid, 3% ethanedithiol, 5% thioanisole,and 2% anisole. The cleaved products are recovered by eitherlyophillization from water or trituration from diethyl ether. Productpurities are estimated from TLC. Selected peptide samples are checked by¹ H NMR to confirm product identity.

Example 2

In this Example (3'-Indolepyruvic acid)-N-biphenylalanine-D-Leu-Asp-OHwas prepared according to the method of Example 1.

Fmoc-N-Asp(Ot-Bu)-MBHA-resin (20 mg) is weighed and transferred to asyringe equipped with a fritted filter. The resin is pre-swollen in 1 mLDMF for 30 minutes. The Fmoc (fluorenylmethyloxycarbonyl) protectinggroup is removed by treatment with 20% piperidine in DMF for 30 minutes.The deprotection solution is removed. The deprotected resin is washedfive times with DMF, five times with MeOH, and then five times with DMF.Fmoc-D-Leu-OH is coupled to the deprotected resin (1 eq) using asolution of Fmoc-D-Leu-OH (3 eq) in 1 mL DMF containing carbodiimide (3eq) and HOBT (hydroxy benzotriazole) (3 eq). A second or third couplingwith solutions of Fmoc-D-Leu-OH may be necessary to achieve couplingefficiencies that pass the Kaiser test. The Fmoc deprotection and aminoacid coupling step are repeated to couple Fmoc-N-(4,4-biphenyl)alanine.The final coupling step uses indolepyruvic acid (5 eq),diisopropylcarbodiimide (5 eq), and HOBT (5 eq) in DMF, and this step isrepeated until the coupling passes the Kaiser test. The completedpeptide sequence on resin is dried under vacuum for at least six hoursand then cleaved by treatment for 2.5 hours with 1 mL of either 95/5trifluoroacetic acid/water or a freshly prepared solution of 90%trifluoroacetic acid, 5% thioanisole, 3% ethanedithiol, and 2% anisole.The cleaved products are recovered by either lyophillization from wateror trituration from diethyl ether. Product purities are estimated fromTLC.

¹ H NMR (400 MHz, d₆ -DMSO): δ 6.5-7.7 (m, 14H), 4.5 (m, 1H), 4.1(m,2H), 3.4(m, 2H), 3 (m, H), 2.7 (m, 1H), 1.1-1.5 (m, 3H), 0.5-0.9 (m,6H).

Example 3

In this example, (3'-Indolepyruvic acid)-N-biphenylalaanine-D-Leu-Asp-OHwas prepared using Chiron Mimotopes Pin Technology

The first amino acid residue Xaa is attached to4-(hydroxymethyl)phenoxyacetamido handle) resin pins (5.7 μmole/pin) bycoupling each pin in 800 μL of coupling solution (100 mM amino acid, 100mM DIC, 10 mM DMAP, 1/4 DMF/CH₂ Cl₂) for two hours. The pins are thenrinsed with a 5 min DMF wash, two 5 min MeOH washes, and 15 minutes ofair drying. Deprotection of the Fmoc group is carried out for 30 minwith 800 μL 20% piperidine in DMF. Repeat pin washings (1 DMF wash, 2MEOH washes, 15 minutes air drying). The second amino acid residue Yaawas coupled (100 mM Yaa, 100 mM DIC, 100 mM HOBT, and bromophenol blueindicator in DMF) until the blue color no longer adheres to the pinsurface. The coupling was repeated as necessary. The rinse cycle andFmoc deprotection washes were then repeated as well. The next aminoacid, Zaa, was coupled by repeating the coupling and washing proceduresfor coupling Yaa, repeating the coupling if necessary. The last residue,indolepyruvic acid is coupled with 15 eq, 100 mM, 15 eq DIC, 15 eq HOBT,and bromophenol blue indicator in DMF. Repeat coupling if necessary.After the last wash, the orange pins were removed from their supportsand cleaved in individual 2 mL plastic centrifuge tubes with 1.5 mL of afreshly prepared solution of 90%, trifluoroacetic acid, 5% thioanisole,3% ethanedithiol, and 2% anisole for 2.5 hours. The pins were removedfrom the tubes and the mixture was blown to near dryness under anitrogen stream. Triturate with Et₂ O and spin down each tube. This stepwas repeated three times per tube. The precipitated peptides werecollected, lyophillized, weighed, and used. Product purity was estimatedby TLC. Initial products were cospotted and checked against authenticsamples obtained in Example 1.

Example 4

Compounds of this invention prepared according to the method of Example1 were tested as follows. The 20S catalytic subunit of the proteasome(also known as the multicatalytic proteinase complex) was purified tohomogeneity from bovine brain according to published methods (Wilk S.and Orlowski, M 1983, 40 842 J.Neurochem). The chymotryptic activity ofthe complex is measured by the increase in fluorescence followingcleavage of the substrate peptidesuccinyl-leucine-leucine-valine-tyrosine-7-amino-4methyl coumarin. Thestandard in vitro assay consists of 2 μg 20S proteasome, 0.1-100 μg/mlproteasome inhibitor in 200 μl 50 mM HEPES, containing 0.1% sodiumdodecyl sulphate, pH 7.5. The proteolytic reaction is initiated by theaddition of 50 μM flourogenic peptide substrate and allowed to progressfor 15 minutes at 37° C. The reaction is terminated by the addition of100 μL of 100 mM acetate buffer, pH 4.0. The rate of proteolysis isdirectly proportional to the amount of liberated aminomethylcoumarinwhich is measured by fluorescent spectroscopy (EX 370 nm, EM 430 nm).

The results of the 20S proteasome inhibitor assays are presented inTable II.

                  TABLE II                                                        ______________________________________                                        IC 50 values for the inhibition of the                                          chymotrypsin-like activity of 20S proteasome.                                       Compound #                                                                              IC.sub.50 μg/mL                                          ______________________________________                                        1             10                                                                2 10                                                                          3 >10                                                                         4 10                                                                          5 >10                                                                         6 >10                                                                         7 >10                                                                         8 >10                                                                         9 >10                                                                         10 >10                                                                        11 >10                                                                        12 >10                                                                        13 >10                                                                        14 >10                                                                        15 10                                                                         16 10                                                                         17 >10                                                                        18 >10                                                                        19 >10                                                                        20 >10                                                                        21 >10                                                                        22 >10                                                                        23 >10                                                                        24 >10                                                                        25 >10                                                                        26 >10                                                                        27 >10                                                                        28 >10                                                                        29 >10                                                                        30 >10                                                                        31 >10                                                                        32 >10                                                                        33 >10                                                                        34 >10                                                                        35 >10                                                                        36 >10                                                                        37 >10                                                                        38 >10                                                                        39 >10                                                                        40 >10                                                                        41 >10                                                                        42 >10                                                                        43 >10                                                                        44 >10                                                                        45 >10                                                                        46 >10                                                                        47 >10                                                                        48 >10                                                                        49 >10                                                                        50 >10                                                                        51 >10                                                                        52 >10                                                                        53 >10                                                                        54 >10                                                                        55 >10                                                                        56 >10                                                                        57 >10                                                                        58 >10                                                                        59 >10                                                                        60 >10                                                                        61 >10                                                                        62 >10                                                                        63 >10                                                                        64 >10                                                                        65 >10                                                                        66 >10                                                                        67 >10                                                                        68 >10                                                                        69 >10                                                                        70 >10                                                                        71 >10                                                                        72 >10                                                                        73 >10                                                                        74 >10                                                                        75 >10                                                                        76 >10                                                                        77 >10                                                                        78 >10                                                                        79 >10                                                                        80 >10                                                                        81 >10                                                                        82 >10                                                                        83 >10                                                                        84 >10                                                                        85 >10                                                                        86 >10                                                                        87 >10                                                                        88 >10                                                                        89 >10                                                                        90 >10                                                                        91 >10                                                                        92 >10                                                                        93 >10                                                                        94 >10                                                                        95 >10                                                                        96 >10                                                                        97 >10                                                                        98 >10                                                                        99 >10                                                                        100 >10                                                                       101 >10                                                                       103 >10                                                                       104 >10                                                                       105 >10                                                                       106 >10                                                                       107 >10                                                                       108 >10                                                                       109 >10                                                                       110 >10                                                                       111 >10                                                                       113 >10                                                                       114 10                                                                        115 10                                                                        116 10                                                                        117 10                                                                        118 10                                                                        119 >10                                                                       120 >10                                                                       121 >10                                                                       122 >10                                                                       123 >10                                                                       124 >10                                                                       125 >10                                                                       126 >10                                                                       127 >10                                                                       128 >10                                                                       129 10                                                                        130 10                                                                        131 10                                                                        132 10                                                                        133 10                                                                        134 >10                                                                       135 >10                                                                       136 >10                                                                       137 >10                                                                       138 >10                                                                       139 >10                                                                       140 >10                                                                       141 >10                                                                       142 >10                                                                       143 >10                                                                       144 10                                                                        145 10                                                                        146 10                                                                        147 10                                                                        148 10                                                                        149 10                                                                        150 >10                                                                       151 >10                                                                       152 >10                                                                       153 >10                                                                       154 >10                                                                       155 >10                                                                       156 >10                                                                       157 >10                                                                       158 >10                                                                       159 >10                                                                       160 >10                                                                       161 >10                                                                       162 >10                                                                       163 >10                                                                       164 >10                                                                       165 >10                                                                       166 >10                                                                       167 >10                                                                       168 >10                                                                       169 >10                                                                       170 >10                                                                       171 >10                                                                       172 >10                                                                       173 >10                                                                       174 5                                                                         175 >10                                                                       176 1                                                                         177 10                                                                        178 >10                                                                       179 >10                                                                       180 5                                                                         181 10                                                                        182 >10                                                                       183 10                                                                        184 >10                                                                       185 5                                                                         186 >10                                                                       187 >10                                                                       188 5                                                                         189 >10                                                                       190 3                                                                         191 3                                                                         192 3                                                                         193 >10                                                                       194 >10                                                                       195 >10                                                                       196 >10                                                                       197 10                                                                        198 >10                                                                       199 >10                                                                       200 >10                                                                       201 >10                                                                       202 >10                                                                       203 >10                                                                       204 >10                                                                       205 >10                                                                       206 >10                                                                       207 10                                                                      ______________________________________                                    

Compounds of this invention prepared according to the method of Example1 were also tested as follows. The 20S catalytic subunit of theproteasome (also known as the multicatalytic proteinase complex) waspurified to homogeneity from bovine brain according to published methods(Wilk S. and Orlowski, M 1983, 40 842 J. Neurochem). The trypticactivity of the complex is measured by the increase in fluorescencefollowing cleavage of the substrate peptideCBZ-D-Ala-Leu-Arg-(7-amino-4-methyl coumarin). The standard in vitroassay consists of 2 μg 20S proteasome, 0.1-1004 g/ml proteasomeinhibitor in 200 ml 50 mM HEPES, containing 0.1% sodium dodecylsulphate, pH 7.5. The proteolytic reaction is initiated by addition of50 mM flurogenic peptide substrate and allowed to progress for 15minutes at 37° C. The reaction is terminated by the addition of 100 mLof 100 mM acetate buffer, pH 4.0. The rate of proteolysis is directlyproportional to the amount of liberated aminomethylcoumarin which ismeasured by fluorescent spectroscopy (EX 370nm, EM 430 nm). Compounds1-207 were tested for tryptic activity inhibition and active asinhibitors at >10 μg/mL.

Example 5

Compounds of this invention prepared according to the method of Example1 were also tested as follows. The 20S catalytic subunit of theproteasome (also known as the multicatalytic proteinase complex) waspurified to homogeneity from bovine brain according to published methods(Wilk S. and Orlowski, M. 1983, 40 842 J. Neurochem). The trypticactivity of the complex is measured by the increase in fluorescenefollowing cleavage of the substrate peptide CBZD-Ala-Leu-Arg-(7-amino-4-methyl coumarin). The standard in vitro assayconsists of 20 μg 20S proteasome, 0.1-100 μg/ml proteasome inhibitor in200 μL 50 mM HEPES, containing 0.1% sodium dodecyl sulphate, pH 7.5. Theproteolytic reaction is initiated by the addition of 5 mM fluorogenicpeptide substrate and allowed to progress for 15 minutes at 37° C. Thereaction is terminated by the addition of 100 μL of 100 mM acetatebuffer, pH 4.0. The rate of proteolysis is directly proportional to theamount of liberated aminomethylcoumarin which is measured by fluorsecentspectroscopy (EX 370 nm, EM 430 nm). Compounds 1-207 were tested fortryptic activity inhibition and were active as inhibitors at >10 μg/mL.

Example 6

Compounds of this invention prepared according to the method of Example1 were also tested as follows. The 20S catalytic subunit of theproteasome (also known as the multicatalytic proteinase complex) waspurified to homogeneity from bovine brain according to published methods(Wilk S. and Orlowski, M. 1983, 40 842 J. Neurochem). The trypticactivity of the complex is measured by the increase in fluorescencefollowing cleavage of the substrate peptide CBZD-Ala-Leu-Glu-(7-amino-4-methyl coumarin). The standard in vitro assayconsists of 2 μg 20S proteasome, 0.1-100 μg/ml proteasome inhibitor in200 ml 50 mM HEPES, containing 0.1% sodium dodecyl sulphate, pH 7.5. Theproteolytic reaction is initiated by the addition of 50 mM fluorogenicpeptide substrate and allowed to progress for 15 minutes at 37° C. Thereaction is terminated by the addition of mL of 100 mM acetate buffer,pH 4.0. The rate of proteolysis is directly proportional to the amountof liberated aminomethylcoumarin which is measured by fluorescentspectroscopy (EX 370 nm, EM 430 nm). Compounds 1-207 were tested forpeptidylglutamyl activity inhibition at >10 μg/mL. Compound 190 wasactive at 5 μg/mL.

What we claim is:
 1. A compound having the following formula: ##STR602##wherein: X₂ is Ar--X₃, wherein X₃ is --CH₂ CO, Ar is indole, R₁ isisobutyl, R₂ is selected from substituted and unsubstituted biphenyl,and X₁ is: ##STR603## wherein R₃ is --CH₂ CO₂ H, and X₄ is --OH.
 2. Thecompound of claim 1 wherein R₂ is biphenylmethane.